Transcription elongation in eucaryotic cells is highly regulated. Numerous elongation factors have been identified, and mutant forms of some factors appear to be involved in disease. The AIDS virus and the hsp70 heat shock gene of Drosophila have served as paradigms for elongation control. Both appear to be negatively regulated. In the case of the AIDS virus, a viral factor called Tat up-regulates transcription of the genome by converting polymerase to a form that resists premature termination. In the case of the hsp70 gene, a polymerase molecule is found stably paused in the promoter proximal region. The GAGA factor, which associates with specific sequences upstream from hsp70 promoter, appears to be involved in establishing the paused state. Transcriptional activation involves heat shock factor, which somehow causes release of the paused polymerase. Several factors, including DSIF and P-TEFb, appear to be involved in elongation control of both hsp70 and the AIDS virus. The overall objectives of this proposal are to understand the basis for the negative regulation of elongation that occurs on the hsp70 promoter prior to heat shock, and how HSF releases the paused polymerase during activation. Insight into these two processes could have significant impact on understanding expression of genes involved in cancer, viral infection, and development. The specific aims are: 1) Test the contributions of the negative elongation factors, DSIF and NELF, and a phosphatase, FCP1, toward promoter proximal pausing in a cell- free transcription system. 2) Analyze the function of DSIF, NELF and FCP1 in living cells. 3) Identify GAGA factor's pausing domain. 4) Identify heat shock factor's releasing domain. Permanganate will be used to monitor for paused polymerase on the hsp70 promoter region in cell-free extracts and in live Drosophila salivary glands. Contributions of DSIF, NELF, and FCP1 in vitro will be assessed by immunodepletion. The role of factors in vivo will be analyzed by several methods including immunofluorescence staining of polytene chromosomes and genomic footprinting analysis of transgenic flies expressing dominant negative forms of the factors and analysis of flies containing potential mutations in these factors. The pausing and releasing domains of GAGA factor and HSF, respectively, will be identified by a transgenic strategy. Parts of each protein will be expressed in flies as fusions with foreign DNA binding domains, and the effect of these fusion proteins on transgenes will be determined by genomic footprinting with permanganate.